The study aimed to analyze the effect of nitrogen and sulfur fertilization on the content and uptake of macronutrients in the dry matter (DM) of spring rye grain. The field experiment was carried out on Cambisols soil (WRB 2015) with a low sulfur content, in south-eastern Poland. The effects of fertilization with N (factor I) 0, 30, 60, 90 kg ha-1 and fertilization with S (factor II) 0, 40 kg ha-1 were investigated. The analysis showed that spring rye responded positively to N and S. N fertilization. Better grain yield was obtained after applying 90 kg N ha-1 (3.684) and adding 40 kg S ha-1 (3.230 t ha-1). In the mentioned combination, the rye grain contained the most macronutrients, with the exception of P, and the uptake of the elements was also the most advantageous. The addition of S in the dose of 40 kg S ha-1 improved the effect of N and the yield of rye grain, in addition to which the content and uptake of macronutrients in the dry matter of rye grain were higher. The best yield of spring rye grain and the content and uptake of all macronutrients, except for potassium (K), in dry matter (DM), were recorded in the 2011 growing season, when the optimal distribution of precipitation was observed. Significant correlations were also found between the grain yield and the content and uptake of all the studied macronutrients, except for the correlation between the grain yield and the K content. Generally, a positive correlation was found between the content and uptake of macronutrients, except for the content of P. Fertilization with N significantly reduced the mass ratios of K+:Ca2+, K+:Mg2+ and the mass and mole ratio of K+:(Ca2++Mg2+), and increased the mass ratio of Ca:P. By contrast, the addition of S significantly reduced the mass ratios of K+:Ca2+ and K+:(Ca2++Mg2+) and increased that of Ca:P.